Purchase this article with an account.
K. Ahn, S. Sohn, C. Kee; Unfolded Protein Response Induced by the Accumulation of Mutant Myocilins in Trabecular Meshwork Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1157.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: : Unfolded protein response (UPR) is an intracellular signaling pathway that leads the upregulation of genes encoding endoplasmic reticulum(ER)-resident chaperones and other protein folding catalysts. The response results in the increased folding capacity of ER, hence often inducing the accumulation of misfolded proteins. The present study was undertaken to determine if the response could also be induced in cultured human trabecular meshwork (TM) cells by the expression of mutant myocilin. Methods: Adenoviral vectors were used to express the desired proteins in TM cells. All proteins were expressed as tagged with GFP in their C-terminus to facilitate detection. TM cells were transduced with adenoviral vectors expressing myocilin with the following mutations; G364V, Q368X, K423E, Y437H, and I477S. After 24 to 72 hours, the induction of two typical end products of UPR, GRP78 and Protein disulfide isomerase (PDI), were analyzed by western blotting. Because UPR is elicited by the overexpression of wild-type proteins or viral infection, parallel experiments were performed with adenoviral vectors expressing wild-type myocilin and stromelysin. Results: : All mutant myocilins were detected exclusively in cell lysates as 83 kDa bands but not in culture media. In cells expressing mutant myocilins, GFP signals of the myocilins increased up to 72 hours in a time-dependent manner, accompanied by the prominent increase in the steady-state levels of GRP78 and PDI. Whereas, in cells expressing wild-type myocilin or stromelysin, the induction of both GRP78 and PDI was not significant, reflecting the fact that UPR induced by mutant myocilins was not merely due to the overexpression of the proteins or the side effect of adenovirus carrying gene for the protein. Conclusions: Mutant myocilins expressed in TM cells accumulated in ER where they provoked UPR. However, little or no myocilin secreted from cells expressing mutant myocilins, suggesting that the relevant folding of the myocilins was rarely achieved even under large amounts of protein chaperones. This fate of mutant myocilins might burden cellular machinery and lead to fundamental threat to living cells.
This PDF is available to Subscribers Only